Intracellular collagen content of SMCs and 3T3s in response to PHD2 WT or cko conditioned medium was measured using CNA35-FITC (Kindly provided by prof. Reutelingsperger, Biochemistry department Maastricht). CNA35-FITC shown to bind to collagen type I, III and IV.18 (link) Samples were analysed using the BD Pathway 855 High Content Bioimager. Data were processed with Attovision and BD Diva software.
SMCs and 3T3s were treated with PHD2 WT or cko conditioned medium for 72 h. Culture medium of SMCs and 3T3s was collected and analysed using Sircol soluble collagen assay as described by the manufacturer (Biocolor, S1000). In comparable subsequent experiments, transcription growth factor (TGF)-β1 was added to the conditioned medium or proteins were heat-inactivated (30 min, 85°C) prior to addition to 3T3 fibroblasts.
SMCs and 3T3s were treated with PHD2 WT or cko conditioned medium for 72 h. Culture medium of SMCs and 3T3s was collected and analysed using Sircol soluble collagen assay as described by the manufacturer (Biocolor, S1000). In comparable subsequent experiments, transcription growth factor (TGF)-β1 was added to the conditioned medium or proteins were heat-inactivated (30 min, 85°C) prior to addition to 3T3 fibroblasts.
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